In recent years there has been increasing attention on phytoestrogens particularly isoflavonoids. Isoflavonoids or isoflavones (as they are also known) are a class of phyto-oestrogens which are found in plants and which are based on a diphenolic ring structure. Due to their structure, it has been documented that they are able to bind to oestrogen receptors on animals including humans. A small subgroup of isoflavones are known to display oestrogenic activity, as well as anti-carcinogenic, antifungal, antiproliferative properties and anti-oxidative effects. These oestrogenic isoflavones (genistein, biochanin, daidzein, glycitein and formononetin) are predominantly found in plants which are members of the Leguminosae family.
Most legumes have been found to contain at least one or more of these oestrogenic isoflavones, with the richest sources being soya beans, lentils, clover, chick peas, alfalfa and other beans. Most human diets contain low to moderate levels of oestrogenic isoflavones. In typical diets in developed Western countries, the dietary intake of the oestrogenic isoflavones is low and often negligible, as legumes are not relied upon strongly as a source of protein, being instead replaced by animal products.
However, the dietary intake of oestrogenic isoflavones from traditional diets of Eastern and developing countries such as India, China and South America is moderate to high, given the fairly high dietary intake of beans including soya beans, kidney beans, lima beans, broad beans, butter beans, chick peas and lentils. The presence of such dietary levels of oestrogenic isoflavones is confirmed by detection of the amounts of the isoflavones daidzein, genistein, glycitein, formononetin and biochanin and their metabolites in human urine. People with high legume intake in their diets excrete substantially higher amounts of isoflavone metabolites in their urine than people with largely omnivorous or low-legume diets.
After ingestion, isoflavones undergo varying degrees of metabolism within the digestive system. The naturally occurring, water soluble glycosidic form of isoflavone undergoes hydrolysis to the aglycone form in the gut, while biochanin and formononetin are demethylated by bacterial fermentation to genistein and daidzein respectively. It appears that the majority of the aglycone isoflavones then undergo fermentation by intestinal bacteria to produce end products including equol, dehydroequol, O-desmethylangolensin (ODMA), dihydrodaidzein, tetra-hydrodaidzein and dihydrogenistein. The isoflavones, their metabolites and derivatives circulate around the body and are mainly excreted in the urine, in which they can then be detected.
As stated above, given the presence of high levels of isoflavones in legumes, particularly soya beans, and the knowledge that the isoflavones are fermented or metabolised by intestinal or bowel bacteria to produce isoflavone metabolites, research has been conducted into microbial fermentations of soybeans and has demonstrated production of metabolites including 6,7,4′-trihydroxyisoflavone (hereinafter called Factor 2) and other polyhydroxylated isoflavonoids.
Traditional Asian food products such as tempeh, tofu, miso etc are foods produced from soybeans by fermentation mainly by fungi of the genus Rhizopus. It has been shown that several bacteria species may also be involved in tempeh production. For traditional tempeh fermentation, the soybeans are cooked, dehulled and soaked overnight. A spontaneous bacterial acidification occurs during this phase. In industrial tempeh fermentation processes, the cooked soybeans are acidified with lactic acid. After the soaking process, the soybeans are cooked again and incubated with microbial inocula for 2 days.
In unfermented soybeans, the isoflavones genistein, daidzein and glycitein predominantly occur as isoflavone glucosides and acylglucosides. It has been shown that during tempeh fermentation, the isoflavone aglycones are liberated from the conjugates and accumulate in the tempeh product. Further findings have shown that during fermentation the isoflavone 6,7,4′-trihydroxyisoflavone (termed “Factor 2” by Gyorgy et al. in Nature (1964) 203. 870-872), also accumulates.
It was previously thought that the fungi of the genus Rhizopus were responsible for the formation of Factor 2 from either daidzein or glycitein. However, subsequent studies on the metabolism of daidzein and glycitein by Klus et al., 1993 showed that isolates of Brevibacterium epidermidis and Micrococcus luteus, which were isolated from Indonesian tempeh samples, readily transform glycitein, forming Factor 2. A third tempeh-derived bacterium, Microbacterium arborescens, metabolized daidzein, producing both Factor 2 and glycitein. More recently, Klus, K. and Barz, W. Arch. Microbiol. 164:428-434, (1995) investigated five other bacterial isolates, which were isolated from tempeh samples containing Factor 2 and were classified as Micrococcus or Arthrobacter strains, for their ability to metabolize daidzein and glycitein by hydroxylation or O-demethylation reactions. Their results show that a number of polyhydroxylated isoflavones were formed, hydroxylated at three or four of positions 6, 7, 8, 3′ and 4′. Of these Factor 2 was the major product produced by most of the microbial strains. The bacterial strains only hydroxylated but did not degrade the substrates namely daidzein or glycitein. The compounds of the present invention were not identified by Klus and Barz, however,
Various polyhydroxylated isoflavones known in the prior art are known to exhibit anti-inflammatory and anti-allergenic activity and to express anticarcinogenic properties due inhibition of protein tyrosine kinases, which play a key role in cellular pathways in tumour cell growth. In in vitro tests, these isoflavones also inhibit the growth of human leukemia (Makishima et al., 1991) and human breast cancer cells (Hirano et al, 1989; Peterson and Barnes, 1991). In essence, the polyhydroxylated isoflavones occurring as dietary factors in fermented soybean products are putative causes of the lower incidence of cancer-related diseases in Asian populations, and have been used in the treatment of a variety of cancers including breast cancer, ovarian cancer, large bowel cancer; and prostatic cancer.
Other therapeutic uses of the oestrogenic isoflavones which have been disclosed include their use as therapeutics for menopausal symptoms and osteoporosis (WO 98/50026, European patent application 0135172, U.S. Pat. No. 5,498,631 in the name of Gorbach et al); pre-menstrual symptoms; Reynauds Syndrome; rheumatic diseases; Buergers Disease; coronary artery spasm; migraine headaches; benign prostatic hypertrophy and hypertension.
As stated above, isoflavonoids are natural plant compounds which possess antitumorigenic properties. Of all oestrogenic isoflavones of which daidzein, genistein, formononetin and biochanin-A are the most well known, it has been shown that individually, genistein is the most potent inhibitor (IC50=25-33 μM) of the proliferation of MCF-7 cells induced by a number of environmental chemicals such as 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane, 5-octylphenol and 4-nonylphenol as demonstrated recently by Verma S P and Goldin B R (Nutrition & Cancer 30(3):232-9. 1998).
The same authors also noted that a mixture of isoflavones was the most potent inhibitor against the induced proliferation. However, as in the case of other research workers they found that genistein, biochanin A, equol and to some extent daidzein at <10 μM can enhance the growth of MCF-7 cells.
There is therefore a need for novel isoflavonoids which can inhibit the proliferation of cancer cells but which do not enhance their growth at low concentrations, and which exhibit other therapeutic properties.